The present invention relates to a liquid (or solution)-phase manufacturing process for preparing the decapeptide Degarelix, its amino-protected precursor, and other useful intermediates. The invention further relates to polypeptides useful in the solution-phase manufacturing process and to the purification of Degarelix itself.

Prostate cancer is a leading cause of morbidity and mortality for men in the industrialised world. Degarelix, also known as FE200486, is a third generation gonadotropin releasing hormone (GnRH) receptor antagonist (a GnRH blocker) that has been developed and recently approved for prostate cancer patients in need of androgen ablation therapy (Doehn et al., Drugs 2006, vol. 9, No. 8, pp. 565-571; WO 09846634). Degarelix acts by immediate and competitive blockade of GnRH receptors in the pituitary and, like other GnRH antagonists, does not cause an initial stimulation of luteinizing hormone production via the hypothalamic-pituitary-gonadal axis, and therefore does not cause testosterone surge or clinical flare (Van Poppel, Cancer Management and Research, 2010:2 39-52; Van Poppel et al., Urology, 2008, 71(6), 1001-1006); James, E.F. et al., Drugs, 2009, 69(14), 1967-1976).

Degarelix is a synthetic linear decapeptide containing seven unnatural amino acids, five of which are D-amino acids. It has ten chiral centers in the back bone of the decapeptide. The amino acid residue at position 5 in the sequence has an additional chiral center in the side-chain substitution giving eleven chiral centers in total. Its CAS registry number is 214766-78-6 (of free base) and it is commercially available under the Trademark Firmagon™. The drug substance is chemically designated as D-Alaninamide, N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-4-[[[(4S)-hexahydro-2,6-dioxo-4-pyrimidinyl]carbonyl]amino]-L-phenylalanyl-4-[(aminocarbonyl)amino]-D-phenylalany-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-prolyl- and is represented by the chemical structure below:

The structure of Degarelix can also be represented as:

• Ac-D-2Nal-D-4Cpa-D-3Pal- Ser-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH₂

For the purposes of describing this invention, each amino acid in Degarelix will be given the shorthand notation as follows:

• AA₁ is D-2Nal, AA₂ is D-4Cpa, AA₃ is D-3Pal, AA₄ is Ser, AA₅ is 4Aph(L-Hor), AA₆ is D-Aph(Cbm), AA₇ is Leu, AA₈ is Lys(iPr), AA₉ is Pro and AA₁₀ is D-Ala.

Thus, as an example, Degarelix can be represented as Ac-AA₁-AA₁₀-NH₂, the tetrapeptide Ac-D-2Nal-D-4Cpa-D-3Pal-Ser can be represented as Ac-AA₁-AA₄ and the hexapeptide 4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH₂ as AA₅-AA₁₀-NH₂.

Degarelix has previously been prepared using Boc-solid phase peptide synthesis (SPPS) methodology as reported in WO 98/46634 and Jiang et al., J. Med. Chem. 2001, 44, 453-467. Basically, Boc-protected D-Ala is first coupled to MBHA resin in dimethylformamide (DMF)/CH₂Cl₂ using diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) as activating or coupling agents. Once D-Ala is coupled to the resin, synthesis proceeds by washing, deblocking and then coupling the next amino acid residue until the decapeptide has been completed. The side chain primary amino groups of 4Aph in the 5-position and of D-4Aph in the 6-position are protected by Fmoc when they are added and modified with L-Hor and Cbm respectively before the next amino acid in the chain is added. This requires the additional steps of first removing the side-chain protection with piperdine, reacting the newly freed amino group on the peptidoresin with tert-butyl isocyanate or L-hydroorotic acid, ensuring that the reaction is complete with a ninhydrin test and then washing the peptidoresin before adding the next amino acid residue (see also Sorbera et al., Drugs of the Future 2006, Vol. 31, No. 9, pp 755-766).

While Boc-SPPS methodology has afforded sufficient quantities of Degarelix until now, the growing demand for this polypeptide means that ever larger quantities are required. Boc-SPPS, which requires HF cleavage, is not suited to large scale industrial synthesis. Indeed, WO 98/46634 mentions that SPPS is only suitable for limited quantities of up to 1 kg while classical peptide solution synthesis, or liquid phase peptide synthesis (LPPS), is preferred for larger quantities of product. WO 98/46634 does not specify how such synthesis should be performed. Further, expense attributable the large excess of coupling reagents, additives, and amino acids required for the SPPS. While the existence of a liquid phase peptide synthesis of Degarelix has been reported [EMEA Report: Assessment Report for Firmagon™ (Degarelix): Doc. Ref. EMEA/CHMP/635761/2008], as of now no details of such a process have been publically disclosed.

WO 97/34923 and WO 99/26964 are International Application Publications which are concerned with liquid phase processes for the preparation of biologically active peptides. WO 99/26964 is particularly concerned with the liquid phase synthesis of decapeptides having activity as GnRH antagonists. WO 99/26964 lists a number of inherent limitations of the SPPS methodology for producing GnRH antagonists including the limited capacity of the resin, the large excess of reagents and amino acids required, as well as the need to protect all reactive side chains such as the hydroxy group in Ser, the aromatic amino groups in Aph and D-Aph, the ε-i-propylamino group in Lys(i-Pr).

International Application Publication No. WO 99/26964 describes a liquid phase process which involves first preparing the central peptide fragments of the 5 and 6 positions of a decapeptide with the side chains fully elaborated and then assembling the peptide through a "4-2-4", "3-3-4" or "3-4-3" fragment assembly pattern. For example, in the preparation of the GnRH antagonist Azaline B, a tetrapeptide is coupled with a hexapeptide to form the desired decapeptide. When the same fragment assembly pattern is attempted for Degarelix, racemisation of the Serine amino acid (AA₄) occurs resulting in about 20% impurity of L-Ser. This impurity carries over into the final decapeptide and is difficult to remove. Furthermore, when preparing the tetrapeptide AA₁-AA₄ by adding the Ser unit to the tripeptide AA₁-AA₃ following the procedure described in WO 99/26964, tetrabutylammonium ions from the hydrolysis of the benzyl ester group could not be removed completely during the subsequent operations and were carried through to the final product. It was further found that in the Degarelix synthesis, the L- hydroorotyl group rearranges to its hydantoinacetyl analogue when L-dihydroorotic acid is coupled with 4Amp to prepare AA₅. These and other problems with the solution-phase synthesis of Degarelix have now been overcome and a new solution-phase polypeptide synthesis of this decapeptide is disclosed herein for the first time.

The problems of SSPS methods for preparing Degarelix and the drawbacks of LLPS methods as described in WO 97/34923 and WO 99/26964 have now been overcome and are the subject of this invention.

In general, this invention relates to a liquid-phase synthesis of the decapeptide Degarelix.

In one aspect, the invention relates to a liquid-phase process for preparing Degarelix having the formula Ac-AA₁-AA₁₀-NH₂: or a pharmaceutically acceptable salt or solvate thereof;
comprising the step of coupling (P₄)Ac-AA₁-AA₄ with (Pε)AA₅-AA₁₀NH₂ or coupling Ac-AA₁-AA₃ with (P₄)(Pε)AA₄-AA₁₀NH₂ in an organic solvent comprising the two peptides, a peptide coupling reagent and an organic amine base dissolved therein wherein Pε is an ε-amino protecting group and P4 is a hydroxyl protecting group or hydrogen, wherein the peptide coupling agent in the case of coupling (P₄)Ac-AA₁-AA₄ with (Pε)AA₅-AA₁₀NH₂ is selected from one or more of o-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexfluorophosphate (BOP),
and the peptides being represented below: to provide a protected Degarelix precursor having the formula (P₄)(Pε)Ac-AA₁AA₁₀-NH₂: and
comprising the step of cleaving the ε-amino protecting group Pe from a Degarelix precursor according to formula (P₄)(Pε)Ac-AA₁-AA₁₀-NH₂ in an organic solvent comprising the precursor and a cleaving agent dissolved therein to provide Degarelix.

P₄ is a hydroxyl-protecting group or hydrogen, preferably, tBu, (ψPro) (i.e. pseudo-proline), or hydrogen. If P4 is a hydroxyl-protecting group, the process also comprises the step of cleaving the hydroxyl-protecting group P₄ from the Degarelix precursor. The protecting group P₄ is preferably selected in such a way that this cleavage step can be carried out

simultaneously with the cleavage of the amino-protecting group Pε. This is for example the case if both P₄ and Pε are BOC.

A further aspect concerns the liquid-phase process for preparing a Degarelix intermediate having the formula (P₄)Ac-AA₁-AA₄: or a pharmaceutically acceptable salt or solvate thereof, comprising the step of hydrolyzing a compound having the formula (P₄)Ac-AA₁-AA₄-R with an alkaline hydroxide, wherein R represents a carboxyl protecting group, preferably C₁-C₄ alkyl or benzyl,
P4 represents hydrogen or a hydroxyl protecting group: and wherein the alkaline hydroxide is LiOH.

A further aspect concerns a process for preparing the compound (P₄)Ac-AA₁-AA₄-R by coupling Ac-AA₁-AA₃ with (P₄)AA₄-R or coupling Ac-AA₁-AA₂ with (P₄)AA₃-AA₄-R, the peptides being represented below

In each of the formulae described above, AA₁ to AA₁₀, P₄ and P_ε have the same meanings as in formula II, and R represents a carboxyl protecting group, preferably C₁-C₄ alkyl or benzyl

In a further aspect, the tetrapeptide (P4)Ac-AA₁-AA₄ is prepared not by liquid phase synthesis, but by solid phase synthesis. This invention thus also relates to a solid-phase process for preparing a Degarelix intermediate having the formula (P4)Ac-AA₁-AA₄: or a pharmaceutically acceptable salt or solvate thereof, comprising the steps:

1. a) reacting (PN)AA2 with (P4)AA₃-AA₄- to provide (P4, PN)AA₂-AA₄-;
2. b) removal of PN from (P4, PN)AA₂-AA₄- to provide (P4)AA₂-AA₄-;
3. c) reacting (PN)AA₁ with (P4)AA₂-AA₄- to provide (P4, PN)AA₁-AA₄-;
4. d) if PN is not acetyl, removal of PN from (P4, PN)AA₁-AA₄- to provide (P4)AA₁-AA₄- and subsequently acetylating (P4)AA₁-AA₄- to provide (P4)Ac-AA₁-AA₄-; and
5. e) cleaving (P4)Ac-AA₁-AA₄- to provide (P4)Ac-AA₁-AA₄.

A further aspect of the invention concerns liquid-phase process for preparing the hexapeptide (Pε)AA₅-AA₁₀NH₂ comprising the coupling of (Pε)AA₆-AA₁₀NH₂ and (P_X)AA₅, wherein P_X is an amino protecting group and AA₅ to AA₁₀ and Pε have the same meaning as above, to provide (P_X)(Pε)AA₅-AA₁₀NH₂, and cleaving Px with TFA to provide (Pε)AA₅-AA₁₀NH₂, the peptides being represented below:

It should be understood that in the process of preparing Degarelix according to this invention, any of the above process steps may be combined. For example, this invention also embodies a process in which (P₄)Ac-AA₁-AA₄ is first prepared from (P₄)Ac-AA₁-AA₄-R according to the third aspect of the invention or according to the fifth aspect of the invention before being coupled with (Pε)AA₅-AA₁₀-NH₂ to form the protected precursor

(P₄)(Pε)Ac-AA₁AA₁₀-NH₂ according to the second aspect of the invention. The precursor (P₄)(Pε)Ac-AA₁-AA₁₀-NH₂ formed by such a process may then be deprotected according to the first aspect of the invention ultimately giving a single process for preparing Degarelix that incorporates the first, second and third or fifth aspects of the invention.

Naturally, any of the purification steps for Degarelix that are described herein may be incorporated into any process in which Degarelix is the final product.

• Figure 1. Liquid phase preparation of the tetrapeptide Ac-AA₁-AA₄
• Figure 2. Preparation of the Pε protected hexapeptide AA₅-AA₁₀NH₂ where Pε is Fmoc.
• Figure 3. Segment condensation and deprotection to yield Degarelix.
• Figure 4: Solid phase preparation of the tetrapeptide Ac-AA₁-AA₄ with a pseudoproline protecting group on AA4.
• Figure 5: Solid phase preparation of the tetrapeptide Ac-AA₁-AA₄ with a tBu protecting group on AA4.
• Figure 6: Solid phase preparation of BOC-protected AA₅-AA₇
• Figure 7: Solid phase preparation of Fmoc-protected AA₈-AA₁₀ NH₂

The present invention will now be described in more detail.

In a first aspect, the present invention relates to a liquid-phase process for preparing Degarelix having the formula Ac-AA₁-AA₁₀-NH₂ or a pharmaceutically acceptable salt or solvate thereof, as described in the claims. The process comprises the step of cleaving an ε-amino protecting group Pε from a Degarelix precursor according to formula (P₄)(Pε)AA₁-AA₁₀ in an organic solution comprising the precursor and a cleaving agent dissolved therein.

In this case, Pe is any side chain protecting group known in the art such as those described in E. Gross & J. Meienhofer, The Peptides: Analysis, Structure, Biology, Vol. 3: Protection of Functional Groups in Peptide Synthesis (Academic Press, N.Y., 1981). Suitable examples include 9-fluorenylmethyloxycarbonyl (Fmoc), CBZ, and substituted CBZ, such as, e.g., p-chlorobenzyloxycarbonyl, p-6-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, and p-methoxybenzyioxycarbonyl, o-chlorobenzyloxycarbonyl, 2,4-dichforobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, and the like; aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), t-amyloxycarbonyl, isopropyloxycarbonyl, 2-(p-biphenylyl)-isopropyloxycarbonyl, and the like; cycloalkyl urethane-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl; allyloxycarbonyl (Alloc). Preferred protecting groups are Fmoc, Boc and Alloc with Fmoc being most preferred.

If required, the hydroxyl group of Ser may also be protected, although this is not preferred. In this case, P₄ is not hydrogen, but a hydroxyl protecting group such as for example, a C₄-C₆ alkyl (e.g. t-butyl, cyclohexyl), trityl, benzyl, a benzyl ether such as p-methoxybenzyl, or other substituted benzyls such as p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, 2,6-dichlorobenzyl, or (ψPro), (pseudoproline). If Ser is protected, particularly preferred is t-butyl, benzyl and 9-fluorenylmethyl ethers, t-butyl being most preferred. P4 is H, tBu, or (ψPro), preferably tBu or (ψPro),

The cleaving agent used to remove the ε-amino protecting group or the Ser hydroxyl protecting group depends on the nature of the protecting group and are well known in the art. In a preferred embodiment, the same cleaving agent is used for both the ε-amino protecting group and the Ser hydroxyl protecting group, if present.

Preferred cleaving agents for the Ser hydroxyl protecting group are:

• trifluoracetic acid (TFA), HCl, or methanesulfonic acid, particularly for t-butyl ether as a protecting group
• H_2/Pd-C, HF, or trifluoromethane sulfonic acid, particularly for benzyl ether as a protecting group, and
• SiCl_4/anisol, particularly for 2-(methylsulfinyl)benzylether as a protecting group;

Preferred cleaving agents for the ε-amino protecting group are:

• trifluoracetic acid (TFA), HCl, or methanesulfonic acid, particularly for t-butyl carbamates as protecting group
• H₂/Pd-C, HF, or trifluoromethane sulfonic acid, particularly for benzyl carbamates as protecting group, and
• Piperidine, DBU and DEA, particularly for Fmoc as protecting group

Preferred solvents include DCM, DMF, NMP, dioxane, EtOH, Neat HF, and TFA.

Particularly preferred are the different cleavage conditions indicated in the following table 1: Protecting group Protected group Cleavage reagent Solvent Abbreviation Name t-Bu t-Butyl ethers and esters -OH and -CO₂H TFA DCM HCl Dioxane Methanesulfonic acid DCM Bzl Benzyl ethers and esters -OH and H₂/Pd-C EtOH/water -CO₂H HF Neat Trifluoromethanesulfonic acid DCM MsOb 4-(Methylsulfinyl)-benzyl ether -OH SiCl₄/anisol TFA Tce 2,2.2-Trichloroethyl esters -CO₂H Zn AcOH/H₂O Cbz or Z Benzyloxycarbonyl NH₂ H₂/Pd-C EtOH/Water/acid HF Neat Trifluoromethanesulfonic acid DCM Boc tert-Butoxy-carbonyl -NH₂ TFA DCM HCl Dioxane Methanesulfonic acid DCM Fmoc 9-Fluorenylmethoxycarbonyl -NH₂ piperidine DMF DBU (1,8-diazabicyclo[5.4.0]-undec-7-ene) DMF DEA (diethylamine) DMF Trt Trityl (Trt) -OH -NH₂ 1% TFA-DCM DCM TBDMS Tert-butyl-dimethyl-silyl -OH TFA ACOH-THF-H₂O (3:1:1), 18h 0.1 MTBAF in THF. THF Cyclohexyl (CHX or CH_X) Cyclohexyl -OH HF or TFSMA Neat HF or DCM Troc 2,2,2-Trichloroethoxycarbonyl -NH₂ Zn AcOH/H₂O

Typically, a cleaving agent such as piperidine is dissolved in an organic solvent such as DMF, NMP under an inert atmosphere such as N₂ or argon and cooled to a temperature between - 20 and 0°C, preferably -10 and -2°C, e.g. about -5°C. The protected intermediate (pε)AA₁-AA₁₀ is added and the reaction mixture is then stirred at a temperature of between -20 and 25°C, preferably -10 and 10°C and more preferably 0 and 5°C. When the protecting group has been removed (preferably the yield is > 95% yield, most preferably > 99%), the crude Degarelix can be precipitated, filtered and then washed with ether. For example, the crude Degarelix can be precipitated by adding it to ether, such as methyl t-butyl ether (MTBE) or DIPE, and stirring for 10 to 30 minutes. The precipitate can then be washed with ether (preferably DIPE). Subsequently, the solid may be taken up in e.g. ethyl acetate and stirred for some time at room temperature. The fine solid obtained may then be filtered, washed (e.g. with ethyl acetate) and dried under vacuum.

It has been found that extended reaction periods are not detrimental to the quality of the reaction and that no significant (< 0.03% yield as determined by HPLC) increase in hydantoin impurity is observed if the reaction is allowed to proceed for up to 24 hours. Furthermore, particularly in the case of piperidine as a cleaving agent, no hydantoin impurity is observed if the reaction is performed in the presence of 5 Vol% water per volume of solvent (e.g. 0.1 ml of water per 2 ml solvent such as DMF) for up to 20 hours. This demonstrates the robustness of this deprotecting reaction for the LPPS of Degarelix.

4+6 coupling and 3+7 coupling

The process of the invention comprises the step of coupling a tetrapeptide intermediate according to formula (P₄)Ac-AA₁-AA₄ with a hexapeptide intermediate according to formula (pε)AA₅-AA₁₀ or the step of coupling a tripeptide intermediate according to formula Ac-AA₁-AA₃ with a heptapeptide intermediate according to formula (P₄)(Pε)AA₄-AA₁₀. In either case, the protecting group Pε may be any ε-amino protecting group as discussed previously. The hydroxyl group of Ser may also be protected if required (i.e., in this case P4 is not hydrogen, but a hydroxyl protecting group),. The coupling reaction is performed in an organic solution where the two peptides, a peptide coupling reagent and an organic amine base are dissolved therein. A peptide coupling additive may also be present.

The organic solvent, peptide coupling reagent, peptide coupling additive and organic amine base may be any of those known in the art of LPPS.

Typical organic solvents are THF, NMP (N-methyl pyrrolidone), DCM, DMF, DMSO, and mixtures thereof.

Typical peptide coupling reagents are one or more of o-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), o-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), o-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-Cl), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PγBroP), iso-butylchloroformate (IBCF), 1,3 dicyclohexylcarbodiimide (DCC), 1,3-diisopropyl-carbodiimide (DIC), 1-(dimethylaminapropyl)-3-ethylcarbadiimide hydrochloride (WSCDI), N-ethoxycarbonyl-2-ethoxy-1,2-dihydraquinoline (EEDQ), isopropylchloroformate (IPCF), 2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA) and 2-succinimido-1,1,3,3-tetramethyluranium tetrafluoroborate (TSTU). Preferred coupling reagents are DIC, HATU, HBTU, and BOP.

Typical peptide coupling additives are 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), 1-hydroxy-1H-benzotriazole (HOBt), 6-chloro-HOBt, and 1-hydroxy-7-azabenzotriazole (HOAt). Particularly preferred are HOBt and HOAt.

Typical organic amine bases are NMM, DIPEA, TEA, and collidine. Particularly preferred is DIPEA.

Particularly preferred is the combined use of HATU, HOAt, and DIPEA. Another preferred embodiment relates to the combined use of DIC, 6-chloro-HOBt, optionally in combination with copper salts.

Surprisingly, it has been found that the choice of organic solvent, peptide coupling reagent/additive and organic amine base has an effect on the yields of the desired products and on the racemisation of the Ser amino acid in the polypeptide backbone.

For instance, while THF, NMP, DCM, DMF and mixtures thereof can be used as solvents for these coupling reactions, the use of DMF, either alone or in a mixture (e.g. with DCM), increases the yield of the desired final product while at the same time reducing any D-Ser impurity. The use of DMF apparently increases the yield and reduces D-Ser impurities. The activation is rapid in a polar solvent such as DMF but slow in a non-polar solvent such as DCM. The HATU/HOAt combination leads to a highly efficient coupling partly because of rapid activation by a base such as DIEA or NMM followed by rapid coupling in the presence of the same base in a polar solvent like DMF. The effect of different solvents is illustrated in Table 2. Entry solvent Coupling Reagent & additive Yield (%) . Purity by HPLC (%) Remarks Product CDEG-1 Impurity (D-ser) 1 THF EDC.HCl 35 62.42 16.29 NMM as a base; CLEU-15 content 26.5% 2 NMP EDC.HCl 35 62.42 16.29 NMM as a base; CLEU-15 content 0.17% 3 DCM-DMF EDC.HCl 55 47.81 10.39 CLEU-15 content 25.54% 4 DMF EDC.HCl 65 72 92 Collidine as a base 5 DMF HATU/HOAt 74 84.4 1.91 3.0 eq. of DIEA is used 6 DMF HATU/HOAt 74 85.3 1.44 3.0 eq. of DIEA is used 7 DMF HATU/HOAt 87 83.65 1.44 8.0 eq. of DIEA is used

The choice of coupling reagent and additive also has a large effect on the yield and degree of racemisation. Coupling reactions using HBTU, HCTU, TBTU, BOP and HATU were performed and found with HBTU and HATU giving the best overall yields. However, it was found that coupling reactions using BOP or HATU as coupling reagent led to much less racemisation of the Ser amino acid.

The addition of a coupling additive significantly improves the yield of the desired polypeptide. In many cases the coupling additive also reduces the degree of racemisation of the Ser amino acid even further thus leading to a product with fewer impurities. Combinations of coupling reagent and additive which increased yield are TBTU/HOAt, HATU/HOAt and HATU/HOBt. Surprisingly, the combination of TBTU/HOAt or HATU/HOAt increased yield while at the same time reducing racemisation, with HATU/HOAt performing best out of all the combinations tested.

The effect of different coupling agents and additives is illustrated in the following Tables 3 and 4. Entry Base Coupling Reagent & additive CDEG-1 Yield (%) Purity by HPLC (%) Remarks CDEG-1 Impurity (D-ser) 1 NMM EDCHCl/HOBt 67 65 20 Racemization is more 2 NMM EDC.HCl/HOBt 67 65 18.9 Racemization is more 3 DIEA TBTU 39 67.93 13.34 1.3% of CLEU-15 remains. 4 DIEA HBTU 53 54.9 20.49 1.9% of CLEU-15 remains 5 DIEA EDC.HCl 51 - - 68.9% of CLEU-15 remains. 6 NMM HBTU 78 61.42 15.16 9.8% of CLEU-15 remains 7 NMM HCTU 60 51.63 15.92 7.32% of CLEU-15 remains 8 NMM TBTU 53 62.78 14.73 3.75% of CLEU-15 remains 9 NMM BOP 51 46.3 8.92 11.93% of CLEU-15 remains 10 NMM HATU 74 65.97 10.62 4.52% of CLEU-15 remains 11 DIEA TBTU/HOAt 98 76.26 6.8 0.14% of CLEU-15 remains 12 DIEA DIC/HOAt - 20.49 1.18 45.9% of CLEU-15 remains 13 DIEA HATU/HOAt 74 83.16 2.9 No unreacted CLEU-15 14 NMM HATU/HOAt 81 64.0 13.32 7.1% of CLEU-15 remains 15 NMM HATU/HOBt 89 65.0 13.14 4.35% of CLEU-15 remains 16 NMM EDC.HCl/HOAt 74 70.38 13.66 No unreacted CLEU-15 17 DIEA HATU/HOAt 79 79.79 2.3 This reaction was repeated in larger scale 18 DIEA TBTU/HOAt 84 75.8 7.2 Reaction was repeated in larger scale 19 DIEA HATU/HOAt 74 84.4 1.91 " 20 DIEA HATU/HOAt 74 85.3 1.44 " 21 DIEA HATU/HOAt 87 84 1.44 " 22 DIEA HATU/HOAt 79 84.5 1.37 " Entry Coupling Reagent Additive Base CDEG-1 Yield (%) Purity by HPLC (%) Remarks CDEG-1 (D-ser) 1 TBTU HOAt DIEA 98 76.26 6.8 0.14% of CLEU-15 remains 2 DIC HOAt DIEA - 20.49 1.18 45.9% of CLEU-15 remains 3 HATU HOAt DIEA 74 83.16 2.9 Completion of reaction 4 I HATU HOAt NMM 80 64.0 13.32 7.1% of CLEU-15 remains 5 HATU HOBt NMM 89 65.0 13.14 4.3% of CLEU-15 remains 6 EDC.HCl HOAt NMM 74 70.38 13.66 No unreacted CLEU-15 7 HATU HOAt DIEA 79 79.79 2.3 Repeat reaction. B TBTU HOAt DIEA 84 75.8 7.2 Repeat reaction 9 HATU HOAt DIEA 74 84.4 1.91 3.0 eq. of base used 10 HATU HOAt DIEA 74 65.3 1.44 3.0 eq. of base used 11 HATU, HOAt DIEA 87 83.65 1.44 3.0 eq. of base used 12 EDC.HCl HOBt NMM 67 65 20 Racemization is more 13 EDC.HCl HOBt NMM 64 65 17.9 Racemization is more 14 EDC.HCl HOBt NMM 67 65 18.9 Racemization is more 15 HOSu NMM - - - Reaction did not proceed

The choice of organic amine base also affects the reaction. For the present invention, NMM and DIEA are preferred as they allow the desired polypeptide to be obtained in the best yields. DIEA is more preferred since this base reduces the degree of Ser racemisation. It has also been found that the amount of base affects the reaction, When a base such as DIEA is used, it was found that the more base present, the lower the yield and higher the degree of racemisation. For example, six equivalents of base (with respect to A51-A10) lead to a twofold increase of the racemisation product as when three equivalents of base are used. Thus, it is preferred to use 1-5 equivalents of base, more preferably 2-4 equivalents of base and most preferably 2.5 to 3.5 equivalents of base in these coupling reactions. The effect of different bases and their amounts is shown in the following tables 5 and 6. Entry Coupling Reagent Additive Base Yeld (%) CDBG-1 Purity by HPLC (%) Remarks CDEC-1 (D-ser) 1 TBTU HOAt DIEA 98 76.26 6.8 0.14% of CLEU-15 remains 2 DIC HOAt DIEA - 20-49 1.18 45.9% of CLEU-15 remains 3 HATU HOAt DIEA 74 83.16 2.9 No CLEU-15 remain 4 HATU HOAt NMM 80 64.0 13.32 7.1% of CLEU-15 remains 5 HATU HOBt NMM 89 65.0 13.14 4.35% of CLEU-15 remains 6 EDCHCl HOAt NMM 74 70.38 13.66 No CLEU-15 remains 7 HATU HOAt DIEA 79 79.79 23 8 TBTU HOAt DIEA 84 75.8 7.2 9 HATU HOAt DIEA 74 84.4 1,91 3.0 eq. of base is used 10 HATU HOAt DIEA 74 85.3 1.44 3.0 eq. of base is used 11 HATU HOAt DIEA 87 83.65 144 3.0 eq. of base is used 12 EDCHCI HOBt Collidine 65 72 9.2 13 EDCHCI HOBt NMM 67 65 20 Racemization is more 14 EDCHCI HOBt NMM 64 65 17.9 Racemization is more 15 EDCHCI HOBt NMM 67 65 18.9 Racemization is more Entry DIEA Qty (eq.) Coupling Reagent & additive CDEB-1 Yield (%) Purity by HPLC (%) Product CDEG-1 Impurity (D-ser) 1 6.0 HATU/HOAt 62 74.19 1.5 2 5.0 HATU/HOAt 69 79.00 3.88 3 4.5 HATU/HOAt 68 84.12 1.18 4 4.0 HATU/HOAt 70 82.64 1.19 5 3.0 HATU/HOAt 74 84.4 1.91 6 3.0 HATU/HOAt 74 85.3 1.44 7 3.0 HATU/HOAt 87 83.65 1.44

The temperature that the coupling reaction is performed also influences the yield and the degree of racemisation of the final product. It was found that a reaction carried out at -15°C gives higher yield, higher purity and less racemisation of the final product than the equivalent reaction carried out at -5°C. Thus, it is preferable to carry out these coupling reactions at temperatures lower than -5°C, preferably lower than -10°C and most preferably at -15°C or lower. The reaction time of these coupling reactions is usually 2-3 hours.

It should be noted that by controlling the reaction temperature and the amount of base added, it is also possible to reduce any hydantoin impurity formation. The hydantoin content is preferably less than 0.5 wt.%, more preferably less than 0.3 wt.%. Thus, it is preferred to use 2.5 to 3.5 equivalents of base at temperatures of -10°C or lower in these reactions.

Finally, the order of addition of the various reagents also plays a role in the final yield, purity and amount of racemisation. If the peptides and coupling additive are first dissolved in the organic solvent before the coupling reagent and the organic amine are added, the overall yield of the desired product is significantly higher. Furthermore, the amount of racemisation is drastically reduced.

        Fragment (P₄)Ac-AA₁-AA₄

The present invention provides different methods for preparing (P₄)Ac-AA₁-AA₄.

The invention relates to a liquid-phase process for preparing a Degarelix intermediate having the formula (P₄)Ac-AA₁-AA₄: or a pharmaceutically acceptable salt or solvate thereof, wherein P₄ is hydrogen or a hydroxyl-protecting group, preferably hydrogen.

When preparing (P₄)Ac-AA₁-AA₄, an ester having the formula (P₄)Ac-AA₁-AA₄-R is first prepared, wherein R is a carboxyl protecting group, such as a benzyl group, preferably however a C₁-C₄ alkyl group. Normally, a benzyl ester of serine is used, the benzyl group being then removed by hydrolysis with tetrabutylammonium hydroxide (see for example WO 99/26964). However, it was found that in the preparation of Degarelix, the tetrabutylammonium ions were not removed completely during subsequent operations and were carried through to the final product. This problem was overcome by using a C₁-C₄ alkyl ester of serine (e.g. serine methyl ester). It was found that the alkyl ester could be easily hydrolyzed using an alkali hydroxide which is LiOH. The yield and quality of the tetrapeptide was not affected by this change and the problem of tetrabutylammonium ion impurities was eliminated.

For example, a compound according to formula (P₄)Ac-AA₁-AA₄-R may be suspended in an organic solvent such as THF and then stirred and cooled to a temperature of between -20 and 5°C, and more preferably -5 and 0°C. An aqueous solution of LiOH is then added to the cooled solution. The aqueous LiOH is added at a rate that maintains the temperature of the cooled solution in the range of -5 to 0°C or below. The solution (oftentimes turbid) is stirred for up to 12 hours, preferably up to 3 hours, before being added, with good stirring, to water with a temperature of 5°C or below, or preferably a mixture of ice and water. Any precipitate at this point is removed by filtration. The pH is then adjusted to pH 4.1-4.3, preferably about 4.2 using any known pH adjusting agent. Preferred is HCl, for example 2M HCl. The precipitate that forms after adjusting the pH is collected by filtration, The precipitate can be further purified by washing it with water, and/or stirring a slurry of it in refluxing MeOH and/or a MeOH/MeCN mixture before collecting it by filtration and then drying it to yield (P₄)Ac-AA₁-AA₄.

A further aspect of the invention concerns a process for preparing the compound (P₄)Ac-AA₁-AA₄-R by coupling Ac-AA₁-AA₃ with (P₄)AA₄-R or coupling Ac-AA₁-AA₂ with (P₄)AA₃-AA₄-R, wherein R is a C₁-C_4-alkyl and P₄ is hydrogen or a hydroxyl-protecting group, preferably hydrogen. For the coupling reaction, essentially the same reagents and conditions as those described above can be used.

In a further aspect, this invention relates to a solid-phase process for preparing a Degarelix intermediate having the formula (P4)Ac-AA₁-AA₄: or a pharmaceutically acceptable salt or solvate thereof, comprising the steps:

1. a) reacting (PN)AA2 with (P4)AA₃-AA₄- to provide (P4, PN)AA₂-AA₄-;
2. b) removal of PN from (P4, PN)AA₂-AA4- to provide (P4)AA₂-AA₄-;
3. c) reacting (PN)AA1 with (P4)AA₂-AA₄- to provide (P4, PN)AA₁-AA₄-;
4. d) if PN is not acetyl, removal of PN from (P4, PN)AA₁-AA₄- to provide (P4)AA₁-AA₄- and subsequently acetylating (P4)AA₁-AA₄- to provide (P4)Ac-AA₁-AA₄-; and
5. e) cleaving (P4)Ac-AA₁-AA₄- to provide (P4)Ac-AA₁-AA₄.

PN is preferably Fmoc, which is preferably removed with piperidine/NMP.

P4 is preferably tBu or (ψPro). Particularly preferred is the combination of (ψPro) for P4 and Fmoc for PN.

Each coupling step is preferably carried out in a manner known per se, preferably however using HATU (or HBTU) and DIPEA and coupling additives.

The starting material (P4)AA₃-AA₄- starting can be prepared by coupling (PN, P4)AA₃-AA₄ to a resin, for example to a 2-ClTrt resin, and then removing PN, or by coupling (PN, P4)AA₄ to to obtain (PN, P4)AA₄-, removing PN, and then reacting (PN)AA₃ with (P4)AA₄- to provide (PN,P4)AA₃-AA₄-, and then removing PN. This is illustrated in Figures 4 and 5.

Suitable resins () include trityl, 2-ClTrt and SASRIN.

in the case P4 is (ψPro) and PN is Fmoc, (PN, P4)AA₃-AA₄ can be prepared following J.Am.Chem.Soc. 1996, 118, 9218-9227. That is, Fmoc protected AA3 is activated; reacted with serine or a salt thereof, and subsequently reacted with acetone or acetone dimethylketal, as illustrated below. Fmoc-D3Pal-Ser((ψ^Me, ^MePro)-OH is particularly preferred as (PN, P4)AA₃-AA₄.         Fragment (P_x)(Pε)AA₅-AA₁₀NH₂

A further aspect of the invention concerns liquid-phase process for preparing the hexapeptide (Pε)AA₅-AA₁₀NH₂ comprising the coupling of (Pε)AA₆-AA₁₀NH₂ and (P_x)AA₅, wherein P_x is an amino protecting group and AA5 to AA10 and Pε have the same meaning as above, to provide (P_x)(Pε)AA₅-AA₁₀NH₂, and cleaving Px with TFA to provide (Pε)AA₅-AA₁₀NH₂.

A further aspect of the invention concerns a liquid-phase process for preparing the hexapeptide (Pε)AA₅-AA₁₀NH₂ by coupling (P5)AA₅-AA₇ with (Pε)AA₈-AA₁₀NH₂ to provide (P5, Pε)AA₅-AA₁₀NH₂ ,and subsequently cleaving P5 to provide (Pε)AA₅-AA₁₀NH₂ (wherein P5 is an amino-protecting group on AA5).

P5 protecting group is preferably BOC . Pε is preferably 9-fluorenylmethyloxycarbonyl (Fmoc). The coupling reaction is preferably carried out in the presence of HATU.

(P5)AA₅-AA₇ and (Pε)AA₈-AA₁₀NH₂ are preferably synthesized by solid phase peptide synthesis, e.g. as illustrated in Figures 6 and 7, respectively. The following combinations of P5 and Pε are preferred: P5 Pε Boc Fmoc Cbz Boc Troc Boc

That is, (P5)AA₅-AA₇OH can be prepared by coupling protected AA7 to a resin; removing the protecting group (e.g. Fmoc); reacting protected AA6 with the obtained product; removing the protecting group (e.g. Fmoc); reacting protected AA5 (e.g. BOC protected) with the obtained product; and cleavage from the resin. This is illustrated in Figure 6.

(Pε)AA₈-AA₁₀NH₂ can be prepared by coupling protected AA10 to a resin, e.g. a Rink amide resin; removing the protecting group (e.g. Fmoc); reacting protected AA9 with the obtained product; removing the protecting group (e.g. Fmoc); reacting protected AA8 (preferably Boc-protected on the alpha-amino group and Fmoc-protected on the side chain) with the obtained product; removing the alpha-amino protecting group; and cleavage from the resin. This is illustrated in Figure 7.

(Pε)AA₈-AA₁₀NH₂ can also be prepared by reacting AA10-NH₂ with protected (e.g. BOC) AA9; removing the protecting group; reacting AA9-AA10 NH₂ with protected AA8 (preferably Boc-protected on the alpha-amino group and Fmoc-protected on the side chain); and removing the protecting group on the alpha-amino group.

In a further aspect, the disclosure relates to the purification of Degarelix. The purification can be carried out in a manner known to the skilled person, e.g. by preparative chromatography.

For example, first purification of Degarelix is achieved with a PLRP-S stationery phase, pH 2.25 using TEAP as a buffer and MeCN (75:25) as mobile phase. Purity of up to 95% can be obtained with this step. If required, a second purification can be carried out using a combination of C8 and C18 columns (e.g. Zorbax) to achieve purity of 99% and above.

The following examples are intended to illustrate a process for the LPPS synthesis of Degarelix. Reference is made to Figures 1 and 2 for the structures of each peptide and polypeptide described herein,

L-Cbz-proline (50.0 g) was dissolved in 2-propanol (500m ≙ 10 V) and the solution was cooled to 15°C. N-Methylmorpholine (25ml ≙ 0.5 V) was then added slowly. After stirring the solution for 15 minutes, 28.49g (1.04 eq.) iso-butyl chloroformate was added dropwise at -15°C. A solution of D-ala-NH₂.HCl (27.48g, 1.1 eq) and NMM (25ml ≙ 0.5 V) in water (250ml ≙ 5 V) was added to the reaction mixture at -15°C. The mixture was stirred for 30 minutes at the same temperature and then warmed to 25°C and stirred for 3-5 hrs. The reaction mixture was quenched by adding ethyl acetate (1000ml ≙ 20 V) and water (10 V) containing NaCl (25g) and NaHCO₃ (25g). The organic layers were separated, washed with water (2×500ml ≙ 10 V) and then dried over sodium sulphate. The organic layer was concentrated to 4 volumes under vacuum below 40°C. The solution was diluted with ethyl acetate (250ml ≙ 5 V) and n-hexane (375ml ≙ 7.5 V) was added dropwise to obtain a white solid. The solid was filtered off and dried to afford the product.
Output: 35.2 g; Yield: 54.7%; [α]₂₅^D: -13.0° [CHCl₃, Literature report: -11.2° (US 5,710, 246)]).
HPLC purity: 99.44%

CLEU-2 (19.5 g) was taken in 2-propanol (129ml ≙ 6.5 V) into a Parr hydrogenation flask and a solution of p-toluenesulphonic acid in water (20ml ≙ 1.0 V) was added to it. 10% Pd/C (5% w/w) was added to the reaction mixture and the mixture then hydrogenated at 40 psi for 2 hrs. When TLC showed the disappearance of the starting material, the catalyst was filtered and washed with 2-propanol (79ml ≙ 4 V) and water (8.75ml ≙ 0.5 V). The filtrate was concentrated under vacuum and stripped off with acetonitrile (4 x 254m1 ≙ 13 V). The residue was taken in a flask and acetonitrile (215 mL ≙ 11 V) was added followed by Boc-Lys(Cbz)-OH (25.5g ≙1.1 equiv) and HOBt (9.9g ≙ 1.2 equiv). The suspension was cooled to - 5°C and NMM (14.95g ≙ 2.45 equiv) was added slowly. Finally, a solution of EDC.HCl (15.3g ≙ 1.3 equiv) in acetonitrile (120ml ≙ 6 V) was added. The reaction mixture was then warmed to 25°C and stirred for 10-12 hrs at the same temperature. The solvent was removed under vacuum below 40°C and diluted with water (120ml ≙ 6 V). The product was extracted with ethyl acetate (878ml ≙ 45 V), water (120ml ≙ 6 V), and 10% sodium carbonate (110ml ≙ 5.7 V). The aqueous layer was extracted with ethyl acetate (2 x 430ml ≙ 2 x 22 V) and the organic layers combined and washed with 10% citric acid solution (2 x 105ml), 10% sodium carbonate solution (2 x 110 ml ≙ 2 x 5 V ), water (120ml ≙ 6 V) and dried over sodium sulphate. The organic layer was concentrated to 10-12 volumes under vacuum below 40°C, stripped off with ethyl acetate (3 x 20 V) and maintained the final 10-12 volumes in each stripping. N-hexane (14 V) was added dropwise to the concentrate mass to get a white solid. The solid was filtered off and dried to afford the product.
Output 29.2 g; Yield: 86.6%; Purity 99.18%, [α]₂₆^D: -26.0° (c 1, CHCl₃)

CLEU-4 (16.3 g) was taken in a mixture of methanol (163ml ≙ 10 V), acetone (22ml ≙ 1.4 V) in a stainless steel Parr hydrogenation flask. 10% Pd/C (10% w/w) was added and the mixture was hydrogenated (60 psi) for 8-12 hrs at 25°C. After the starting material had disappeared (TLC), the catalyst was filtered through celite and was washed with methanol (163ml ≙ 10 V). The filtrate was concentrated under vacuum below 40°C and the residue was stripped off with ethyl acetate (3x 143ml ≙ 3 x 9 V). The residue was then taken in ethyl acetate (51ml ≙ 3 V) and n-hexane (20.4ml ≙ 1.25 V) added. The mixture was stirred for 2-3 hrs to obtain a free solid. The solid was filtered, washed with n-hexane (38ml ≙ 2 V), and dried under vacuum below 40° C.
Output: 12.3 g; Yield: 90.8%; Purity: 98.9%

CLEU-5 (12.2 g) was added to THF (40ml ≙ 3.3 V) and the mixture was cooled to 0°C. 10% sodium carbonate solution (34ml ≙ 2.7 V) was added to the mixture over 20 minutes. Fmoc-Cl (8.63g ≙ 1.2 equiv) in THF (12.2ml ≙ 1.0 V) was then added slowly over 15-20 minutes at 0°C. The reaction mixture was stirred for 1 hr at same temperature and diluted with water (134.2ml ≙ 11 V). The product was extracted with ethyl acetate (269ml ≙ 22 V). The organic layer was washed with water (134.2ml ≙ 11 V), 10% citric acid solution (2 x 134ml ≙ 2×11 V) and water (134.2ml ≙ 11 V). The organic layer was concentrated under vacuum below 40°C and the crude product was purified by column chromatography.
Output: 13.3 g; Yield: 73.2%; Purity: 98.2%

CLEU-6 (9.0 g) was charged to a TFA (61ml ≙ 6.75 V) and m-cresol (0.61 ml) solution at -5°C. The reaction mixture was stirred for 2 hrs at 0°C and then concentrated under vacuum below 35°C. Traces of TFA were removed by co-distillation with toluene (2 x 45ml). The product was crystallized from a mixture of MTBE (9ml ≙ 1 V), and DIPE (90ml ≙ 10 V). The solid was filtered off under nitrogen, washed with DIPE (180ml ≙ 20 V) and dried under vacuum to get pure CLEU-7.
Output: 8.8 g; Yield: 90.4%; Purity: 98.3%.
Comment: CLEU-7 material is hygroscopic in nature and thus should be handled with care.

CLEU-7 (8.5 g) was taken in acetonitrile (85ml ≙ 10 V). Boc-Leu-OH (3.12g ≙ 1.1, equiv), HOBt (2.31g ≙ 1.39 equiv) and NMM (1.4ml ≙ 1.03 equiv) were added to the solution. The solution was cooled to -2°C and treated with NMM (1.4m ≙ 1.03 equiv) and EDC.HCl (2.58g ≙ 1.1 equiv). The reaction mixture was stirred for 2-3 hrs at 0°C and the solvent was removed by distillation under vacuum. 10% citric acid (85ml ≙ 10 V) and ethyl acetate (213ml ≙ 25 V) were added to the residue. The organic layer was separated and washed with 10% citric acid solution (2 x 85ml ≙ 2 x 10.0 V), DM water (85ml ≙ 10 V), and 5 % sodium bicarbonate solution (3 x 85ml ≙ 3 x 10.0 V) and again with DM water (85ml ≙ 10 V). Finally, the organic layer was dried over sodium sulphate and concentrated under vacuum below 35°C to obtain the crude product. The crude product was crystallized from MTBE (68ml ≙ 8 V) and n-hexane (34ml ≙ 4 V). The solid was dried under vacuum at below 35°C.
Output: 7.8 g; Yield: 81.2%; Purity: 97.5%.

CLEU-8 (7.5 g) was charged to a TFA (54ml ≙ 7.2 V) and m-cresol (0.2.7 ml) solution at -5°C. The reaction mixture was stirred for 2.0 hrs at 0°C and then concentrated under vacuum below 35°C. Traces of TFA were removed by co-distillation with toluene (2 x 38ml the product was crystallized from MTBE (75ml ≙ 10 V) and n-hexane (413ml ≙ 15 V). The solid was filtered under nitrogen, washed with n-hexane (37.5ml ≙ 5 V), and dried under vacuum to yield pure CLEU-9.
Output: 7.2 g; Yield: 94.8%; Purity: 97.2%

CLEU-9 (10.5 g) was taken in acetonitrile (158ml ≙ 15 V) under nitrogen atmosphere. CMAP-5A (4.2 g ≙ 1.0 equiv), HOBt (2.11g ≙ 1.2 equiv) were added into the suspension and the mixture was cooled to 0°C. EDC.HCl (2.73g ≙ 1.1 equiv) was added into the suspension followed by slow addition of NMM (1.38g ≙ 1.05 equiv). The reaction mixture was stirred for 0.5 hr at 0°C, warmed to ambient conditions and then stirring was continued for 3 hrs. The solvent was removed under vacuum below 35°C and the residue was taken in a mixture of 10% citric acid (105ml ≙ 10 V) and ethyl acetate (263ml ≙ 25 V). The organic layer was separated and washed with 10% citric acid solution (105ml ≙ 10 V), DM water (105ml ≙ 10 V), 5% sodium bicarbonate solution (3 x 105ml ≙ 3 x 10 V), and DM water (105ml ≙ 10 V). The organic layer was dried over sodium sulphate and concentrated under vacuum below 35°C. The crude product was precipitated with n-hexane (84ml ≙ 8 V), washed with n-hexane (2 x 84ml ≙ 2 x 8 V), and dried under vacuum at 35°C.
Output: 10.5 g; Yield: 80.7%; Purity: 92.1%

CLEU-12 (10.5 g) was charged to a TFA (78.8ml ≙ 7.5 V) and m-cresol (0.4 ml) solution at-5°C. The reaction mixture was stirred for 2hrs at 0°C and then concentrated under vacuum below 35°C. Traces of TFA were removed by co-distillation with toluene (2 x 52ml). The residue was taken up in ethyl acetate (105ml ≙ 10 V) and n-hexane (158ml ≙ 15 V) is added to precipitate the product. The solid was filtered off under nitrogen and washed with n-hexane (53ml ≙ 5 V). The compound was dried under vacuum at 35°C.
Output: 9.6 g; Yield: 90.5%; Purity: 91.9%.

CLEU-13 (9.5 g) was dissolved in DMF (9Sml ≙ 10 V) under a nitrogen atmosphere. NMM (1.0g ≙ 1.05 equiv), CSER-2 (3.95g ≙ 1.0 equiv) and HOBt (1.4g ≙ 1.1 equiv) were added to the solution and the reaction mass was cooled to 0°C. EDC.HCl (2.0g ≙ 1.1 equiv) and NMM (1.0g ≙ 1.05 equiv) were added subsequently into the mixture. The reaction mass was stirred for 5 hrs at 0°C and then poured into ice cooled water (950ml ≙ 100 V) and stirred for 30 minutes. The precipitated solid was filtered off and washed with water (20 V), 10% citric acid (10 V), again with water (190ml ≙ 20 V), 5% NaHCO₃ solution (95ml ≙ 10 V), and water (95ml ≙ 10 V). The product was dried under vacuum below 35°C.
Output: 10.3 g; Yield: 84.40%; Purity: 90.4%.

CLEU-14 (10.0 g) was charged to a TFA (75ml ≙ 7.5g) and m-cresol (0.37 ml) solution at -5°C. The reaction mixture was stirred for 2 hrs at 0°C and then concentrated under vacuum below 35°C. Traces of TFA.were removed by co-evaporation with toluene (2 x 50ml). The residue was taken up in ethyl acetate (100ml ≙ 10 V) and n-hexane (60ml ≙ 6 V) was added to precipitate the product. The solid was filtered off under nitrogen, washed with n-hexane (2 x 30 ml ≙ 6 V) and then dried under vacuum at 35°C.
Output: 9.6 g; Yield: 95.0%; Purity: 91.5%.

To a stirred solution of L-hydroorotic acid (23.4 g, 148 mmol) and N-hydroxysuccinimide (18.14g ≙ 1.1 equiv) in dry DMF (585 ml ≙ 2S V) was added DIC (20.5g ≙ 1.1 equi) with external ice water cooling. The reaction mixture was stirred at room temperature for 13-14 hrs. The precipitate was filtered off and the filtrate was evaporated. The oily residue was washed with diisopropyl ether (94ml ≙ 4 V) and dissolved in dry DMF (293ml ≙ 12.5 V). N-Boc-L-4-aminophenylalanine (41.5 g ≙ 1.0 equiv) was added to the above solution. DIEA (22,97g ≙ 1.2 equiv) was added at 0°C and the reaction mixture was stirred for 22 hrs and the solvent then evaporated. The residue was mixed with water (702ml ≙ 30 V) and the pH of the resulting suspension was adjusted to 9.0 with saturated sodium bicarbonate solution. The precipitate of diisopropylurea was filtered off and the filtrate was washed with ethyl acetate (70.2ml ≙ 3 V). The aqueous layer was acidified to pH 2.5 with 6 N HCl and the resulting precipitate collected by filtration. The product was obtained as a yellow solid.
Output: 40.0 g; Yield 64%; mp-270°C, [α]₂₅^D = +62.5 (c 1.0, 1 % NaHCO₃); Purity 95%.

Fragment-A (20.0 g) (purchased from Chirotech, UK) was dissolved in DMF (300ml ≙ 15 V) at 30-35°C and then cooled -10° C to 5°C wherein HOBt (5.06g ≙ 1.1 equiv) was added to the mixture, stirred for 30 minutes before L-serine methyl ester (5.29g ≙ 1.0 equiv) was added to the suspension. The mixture was stirred for 30 minutes, treated with NMM (7.23g ≙ 2.1 equiv) and then stirred for 30 minutes. EDC.HCl (7.18g ≙ 1.1 equiv) was then added to the suspension and the reaction mixture was stirred for 5 hrs at -5°C to 0°C. The reaction mixture was poured into chilled DM water (1.5l or 1500ml ≙ 75 V) and stirred for 30 minutes. The product was precipitated by stirring at 0-5°C. After 1 hr the resulting solid was collected by filtration. The filter cake was washed with water (200ml ≙ 10 V), 10% citric acid (200ml ≙ 10 V), again with water (200ml ≙ 10 V), 5% NaHCO₃ solution (200ml ≙ 10 V), water (200m! ≙ 10 V) and then the solid was dried under vacuum for 4 hrs. The product was slurried in methanol (300ml ≙ 15 V) and stirred for 1 hr. The suspension was filtered and the cake washed with methanol (100ml ≙ 5 V) then dried under vacuum at 30-35°C to a constant weight.
Output: 22 g; Yield: 93.8%.

CBBC 2B (20.0 g) was suspended in THF (500ml ≙ 25 V) and then stirred and cooled to -5°C to 0°C. To the cooled solution was added an aqueous solution of lithium hydroxide (3.65g ≙ 3.0 equiv) at such a rate that the reaction temperature was maintained at between -5°C and 0°C (about 30 mins). The solid dissolved after the base was added to afford a slightly turbid solution. Stirring was continued for another 2 hrs at below -5°C to 0°C. After 3 hrs the turbid reaction mixture was added slowly, with good stirring, into ice/water (700 ml ≙ 35 V) at 5°C whereby any undissolved particles were filtered under vacuum. While stirring, the pH was adjusted to 4.2 using 2M HCl (≈40ml ≙ ≈2 V). The thick white precipitate was collected by filtration and the damp cake washed with 200ml ≙ 10 V of water, air dried under vacuum briefly, slurried in 400ml ≙ 20 V of methanol and then stirred under reflux. The suspension was filtered and the damp cake washed again with 200ml 10 V of methanol then dried under vacuum. The wet cake was then taken in methanol (400ml ≙ 20 V) and acetonitrile (200ml ≙ 10 V) and stirred under reflux. The suspension was filtered hot and the damp cake washed with 200ml ≙ 10 V of methanol then dried under vacuum. The product was dried under vacuum at 35°C to a constant weight to afford the tetrapeptide acid Ac-(AA₁-AA₄).
Output: 12.0 g; Yield: 62.0 %; Purity: 97.1%.

CLEU-15 (4.3 g), CBBC-3A (2.24g ≙ 1.0 equiv), and HOAt (0.56g ≙ 1.2 equiv) were charged into an RBF containing DMF (^∼26ml ≙ 6 V). The mixture was stirred for 15-30 minutes to yield a clear solution at 25°C and then treated with DIPEA (1.71g ≙ 4.0 equiv) The reaction was then cooled to -3.5°C and HATU (1.57g ≙ 1.25 equiv) was added to the mixture. The reaction mixture was stirred for 2 hrs at -10°C, warmed to 20°C and then stirred for 1 hr at the same temperature. The reaction mass was added to 10% citric acid solution (270ml ≙ 60 V) and stirred for 30 minutes at 10°C. The precipitated solid was filtered, washed with water (270ml ≙ 30 V), 5% NaHCO₃ solution (130ml ≙ 30 V) and again with water (270ml ≙ 60 V). The solid was dried under vacuum at 35°C.
Output: 4.0 g; Yield: 65.1%; Purity 79.25%.

20% piperidine in DMF (25ml ≙ 5 V) was charged into an RBF under a nitrogen atmosphere. The solution was cooled to -5°C and CDEG-1 (5.0 g) was added. The reaction mixture was stirred for 45 minutes at 0°C. The reaction mixture was poured into DIPE (250ml ≙ 50 V) and then stirred for 15 minutes. The precipitated solid was filtered off under nitrogen and washed with DIPE (50ml ≙ 10 V). The solid was then taken up in ethyl acetate (125ml ≙ 25 V) and stirred for 1 hr at 25°C. The fine solid obtained was filtered, washed with ethyl acetate (50ml ≙ 10 V), and then dried under vacuum.
Output: 4.7 g; Yield: Quantitative, Purity: 87.6%, D-Ser impurity 1.5%, hydantoin impurity 0.16%.
HPLC condition for CDEG:

• Column: YMC basic (250mm x 3.0 mm), 5µ
• Mobile phase A: 0.1% TFA in ACN
• Mobile phase B: 0.1% TFA in H₂O
• Wave length: 226 nm, Diluent: Mobile phase A: M.P.B =27:73
• Column tem: 50°C, Inject.vol. 50 µl
• Gradient T/%A = 0/73, 18/70, 41/30, 43/73, 50/73
• Flow rate 0.5ml/min